Download QIAamp® Viral RNA Mini Handbook

Second
Third Edition
Edition
December
June 2005
2012
QIAamp® Viral RNA Mini Handbook
For purification of viral RNA from plasma, serum, cell-free
body fluids, and cell-culture supernatants
Sample & Assay Technologies
QIAGEN Sample and Assay Technologies
QIAGEN is the leading provider of innovative sample and assay technologies, enabling
the isolation and detection of contents of any biological sample. Our advanced,
high-quality products and services ensure success from sample to result.
QIAGEN sets standards in:
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Purification of DNA, RNA, and proteins
Nucleic acid and protein assays
microRNA research and RNAi
Automation of sample and assay technologies
Our mission is to enable you to achieve outstanding success and breakthroughs. For
more information, visit www.qiagen.com.
Contents
Kit Contents
4
Storage
4
Intended Use
5
Safety Information
5
Quality Control
5
Introduction
6
Principle
6
Procedure
6
Cellular DNA contamination
8
Warnings and precautions
9
Sample volumes
9
Lysis
9
Carrier RNA
10
Addition of internal controls
10
Spin and vacuum procedures
11
Automated viral RNA purification on the QIAcube
11
Determination of yield
11
Determination of viral RNA length
11
Equipment and Reagents to Be Supplied by User
13
Important Notes
14
Preparation of reagents
14
Handling of QIAamp Mini columns
17
Handling guidelines for the QIAvac 24 Plus
18
Centrifugation
21
Protocols
Purification of Viral RNA (Spin Protocol)
22
Purification of Viral RNA (Vacuum Protocol)
25
Sample Concentration
29
Purification of Cellular, Bacterial, or Viral DNA from Urine
30
Troubleshooting Guide
31
Appendix
35
Ordering Information
37
QIAamp Viral RNA Mini Handbook 06/2012
3
Kit Contents
QIAamp Viral RNA Kit
(50)
(250)
52904
52906
Number of preps
50
250
QIAamp Mini Spin Columns
50
250
200
1000
Buffer AVL*
31 ml
5 x 31 ml
Buffer AW1* (concentrate)
19 ml
98 ml
Buffer AW2† (concentrate)
13 ml
66 ml
3 x 2 ml
10 x 2 ml
310 µg
5 x 310 µg
1
1
Catalog no.
Collection Tubes (2 ml)
Buffer AVE
†
Carrier RNA (poly A)
Selection Guide
* Contains chaotropic salt which is an irritant. Not compatible with disinfecting reagents that contain bleach.
See page 5 for safety information.
†
Contains sodium azide as a preservative.
Storage
QIAamp Mini spin columns should be stored dry at room temperature (15–25°C);
storage at higher temperatures should be avoided. All solutions should be stored at
room temperature unless otherwise stated. QIAamp Mini spin columns and all buffers
and reagents can be stored under these conditions until the expiration date on the kit
box without showing any reduction in performance.
Lyophilized carrier RNA can be stored at room temperature until the expiration date
on the kit box. Carrier RNA should be dissolved in Buffer AVE; dissolved carrier RNA
should be immediately added to Buffer AVL as described on page 14. This solution
should be prepared fresh, and is stable at 2–8°C for up to 48 hours. Buffer
AVL–carrier RNA develops a precipitate when stored at 2–8°C that must be
redissolved by warming at 80°C before use. Unused portions of carrier RNA dissolved
in Buffer AVE should be frozen in aliquots at –20°C. Do not freeze–thaw the aliquots
of carrier RNA more than 3 times.
DO NOT warm Buffer AVL–carrier RNA solution more than 6 times. DO NOT incubate
at 80°C for more than 5 min. Frequent warming and extended incubation will cause
degradation of the carrier RNA, leading to reduced recovery of viral RNA and
eventually false negative RT-PCR results, particularly when low-titer samples are used.
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QIAamp Viral RNA Mini Handbook 06/2012
Intended Use
The QIAamp Viral RNA Mini Kit is intended for molecular biology applications. This
product is not intended for the diagnosis, prevention, or treatment of a disease.
All due care and attention should be exercised in the handling of the products. We
recommend all users of QIAGEN® products to adhere to the NIH guidelines that have
been developed for recombinant DNA experiments, or to other applicable guidelines.
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves,
and protective goggles. For more information, please consult the appropriate safety data
sheets (SDSs). These are available online in convenient and compact PDF format at
www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN
kit and kit component.
CAUTION: DO NOT add bleach or acidic solutions directly to the
sample preparation waste.
Buffers AVL and AW1 contain guanidine salts, which can form highly reactive compounds
when combined with bleach. If liquid containing these buffers is spilt, clean with a suitable laboratory detergent and water. If the spilt liquid contains potentially infectious
agents, clean the affected area first with laboratory detergent and water, and then with
1% (v/v) sodium hypochlorite.
24-hour emergency information
Emergency medical information in English, French, and German can be obtained 24
hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of
the QIAamp Viral RNA Mini Kit is tested against predetermined specifications to ensure
consistent product quality.
QIAamp Viral RNA Mini Handbook 06/2012
5
Introduction
Please take a few moments to read this handbook carefully before beginning your
preparation. The “Important Notes“ on page 14, and the comments within the
QIAamp Viral RNA Mini protocols, beginning on page 22, are particularly valuable.
Principle
QIAamp Viral RNA Mini Kits provide the fastest and easiest way to purify viral RNA
for reliable use in amplification technologies. Viral RNA can be purified from plasma
(treated with anticoagulants other than heparin), serum, and other cell-free body fluids.
Samples may be fresh or frozen, but if frozen, should not be thawed more than once.
Repeated freeze–thawing of plasma samples will lead to reduced viral titers and
should be avoided for optimal sensitivity. Cryoprecipitates accumulate when samples
are subjected to repeated freeze–thaw cycles. This may lead to clogging of the
QIAamp membrane when using the vacuum protocol.
QIAamp Viral RNA Mini Kits are for general use and can be used for isolation of viral
RNA from a wide variety of viruses, but performance can not be guaranteed for every
virus.
Procedure
QIAamp Viral RNA Mini Kits represent a well established technology for general-use
viral RNA preparation. The kit combines the selective binding properties of a silicabased membrane with the speed of microspin or vacuum technology and is highly
suited for simultaneous processing of multiple samples. QIAamp Viral RNA spin
protocols can be fully automated on the QIAcube®. The sample is first lysed under
highly denaturing conditions to inactivate RNases and to ensure isolation of intact viral
RNA. Buffering conditions are then adjusted to provide optimum binding of the RNA
to the QIAamp membrane, and the sample is loaded onto the QIAamp Mini spin
column. The RNA binds to the membrane, and contaminants are efficiently washed
away in two steps using two different wash buffers. High-quality RNA is eluted in a
special RNase-free buffer, ready for direct use or safe storage. The purified RNA is
free of protein, nucleases, and other contaminants and inhibitors. The special QIAamp
membrane guarantees extremely high recovery of pure, intact RNA in just twenty
minutes without the use of phenol/chloroform extraction or alcohol precipitation. All
buffers and reagents are guaranteed to be RNase-free.
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QIAamp Viral RNA Mini Handbook 06/2012
QIAamp Viral RNA
Mini Spin Procedure
QIAamp Viral RNA
Mini Vacuum Procedure
Sample
Sample
Lyse
Bind
Vacuum
Fully automatable on the QIAcube
Wash
(Buffer AW1)
Vacuum
Wash
(Buffer AW2)
Vacuum
Elute
Pure viral nucleic acid
QIAamp Viral RNA Mini Handbook 06/2012
7
Adsorption to the QIAamp membrane
The buffering conditions of the lysate must be adjusted to provide optimum binding
conditions for the viral RNA before loading the sample onto the QIAamp Mini column.
Viral RNA is adsorbed onto the QIAamp silica membrane during two brief
centrifugation steps or by vacuum. Salt and pH conditions in the lysate ensure that
protein and other contaminants, which can inhibit downstream enzymatic reactions,
are not retained on the QIAamp membrane. If the initial sample volume is larger than
140 µl, it will be necessary to load the lysate onto the QIAamp Mini column in several
steps.
Removal of residual contaminants
Viral RNA, bound to the QIAamp membrane, is washed free of contaminants during
two short centrifugation or vacuum steps. The use of two different wash buffers, AW1
and AW2, significantly improves the purity of the eluted RNA. Optimized wash
conditions ensure complete removal of any residual contaminants without affecting
RNA binding.
Elution with Buffer AVE
Buffer AVE is RNase-free water that contains 0.04% sodium azide to prevent microbial
growth and subsequent contamination with RNases. Sodium azide affects
spectrophotometric absorbance readings between 220 and 280 nm but has no effect
on downstream applications, such as RT-PCR. Should you wish to determine the purity
of the eluted RNA, elution with RNase-free water instead of Buffer AVE is recommended.
Cellular DNA contamination
The QIAamp Viral RNA Mini Kit is not designed to separate viral RNA from cellular
DNA, and both will be purified in parallel if present in the sample. To avoid
copurification of cellular DNA, the use of cell-free body fluids for preparation of viral
RNA is recommended. Samples containing cells, such as cerebrospinal fluid, bone
marrow, urine, and most swabs, should first be filtered, or centrifuged for 10 minutes
at 1500 x g and the supernatant used. If RNA and DNA have been isolated in
parallel, the eluate can be DNase digested using RNase-free DNase, followed by heat
treatment (15 min, 70°C) to inactivate the DNase.
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QIAamp Viral RNA Mini Handbook 06/2012
Warnings and precautions
RNA is extremely sensitive to RNases and should always be prepared with due care.
Hands and dust particles may carry bacteria and molds and are the most common
sources of RNase contamination. Please read “Handling RNA” in the appendix
(page 35) of this handbook before starting.
PCR should always be carried out using good laboratory practices. Accordingly, a
PCR laboratory should always be divided into three areas: an area for preparation of
reagents, an area for preparation of samples, and an area for amplification and
detection. Due to the high sensitivity of PCR, it is absolutely necessary that all reagents
remain pure and uncontaminated, and should be monitored carefully and routinely.
Contaminated reagents must be discarded.
Sample volumes
QIAamp Mini columns can bind RNA greater than 200 nucleotides in length. Actual
yield will depend on sample size, sample storage, and virus titer. The procedure is
optimized for use with 140 µl samples, but samples up to 280 µl can be used. Small
samples should be adjusted to 140 µl with phosphate-buffered saline (PBS) before
loading, and samples with a low viral titer should be concentrated to 140 µl before
processing. For samples larger than 140 µl, the amount of lysis buffer and other
reagents added to the sample before loading must be increased proportionally, but
the amounts of Buffers AW1 and AW2 used in the wash steps usually do not need to
be increased. If the initial sample volume is increased, application of the lysed sample
to the QIAamp Mini column will require multiple loading steps. There is no danger of
overloading the QIAamp Mini column, and the quality of the purified RNA will be
unaffected. For volumes greater than 560 µl, concentration of the sample is
recommended. See “Protocol: Sample Concentration,” page 29.
Lysis
The sample is first lysed under the highly denaturing conditions provided by Buffer AVL
to inactivate RNases and to ensure isolation of intact viral RNA. Carrier RNA, added
to Buffer AVL, improves the binding of viral RNA to the QIAamp membrane especially
in the case of low-titer samples, and limits possible degradation of the viral RNA due
to any residual RNase activity.
QIAamp Viral RNA Mini Handbook 06/2012
9
Carrier RNA
Carrier RNA serves two purposes. Firstly, it enhances binding of viral nucleic acids to
the QIAamp Mini membrane, especially if there are very few target molecules in the
sample. Secondly, the addition of large amounts of carrier RNA reduces the chance
of viral RNA degradation in the rare event that RNase molecules escape denaturation
by the chaotropic salts and detergent in Buffer AVL. If carrier RNA is not added to
Buffer AVL this may lead to reduced viral RNA recovery.
The amount of lyophilized carrier RNA provided is sufficient for the volume of Buffer
AVL supplied with the kit. The concentration of carrier RNA has been adjusted so that
the QIAamp Viral RNA Mini Kit can be used as a generic purification system
compatible with many different amplification systems and is suitable for a wide range
of RNA viruses.
Different amplification systems vary in efficiency depending on the total amount of
nucleic acid present in the reaction. Eluates from this kit contain both viral nucleic acids
and carrier RNA, and amounts of carrier RNA will greatly exceed amounts of viral
nucleic acids. Calculations of how much eluate to add to downstream amplifications
should therefore be based on the amount of carrier RNA added. To obtain the highest
levels of sensitivity in amplification reactions, it may be necessary to adjust the amount
of carrier RNA added to Buffer AVL.
Addition of internal controls
Using the QIAamp Viral RNA Mini protocols in combination with commercially
available amplification systems may require the introduction of an internal control into
the purification procedure. Internal control RNA or DNA should be added together
with the carrier RNA to the lysis buffer. For optimal purification efficiency, internal
control molecules should be longer than 200 nucleotides, as smaller molecules are not
efficiently recovered.
Refer to the manufacturer’s instructions to determine the optimal concentration. Using
a concentration other than that recommended may reduce amplification efficiency.
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QIAamp Viral RNA Mini Handbook 06/2012
Spin and vacuum procedures
The QIAamp Viral RNA Mini purification procedure is carried out in three steps using
QIAamp Mini columns in a standard microcentrifuge, on a vacuum manifold, or on the
QIAcube. The procedures are designed to ensure that there is no sample-to-sample
cross-contamination and allow safe handling of potentially infectious samples.
QIAamp Mini columns fit into most standard microcentrifuge tubes. In the spin protocol,
due to the volume of filtrate, 2 ml collection tubes (provided) are required to support the
QIAamp Mini column during loading and wash steps. For the vacuum protocol, a vacuum
manifold (QIAvac 24 Plus or equivalent see page 18) and a vacuum pump capable of
producing a vacuum of –800 to –900 mbar (e.g., Vacuum Pump) are required.
Eluted RNA can be collected in standard 1.5 ml microcentrifuge tubes (not provided).
These tubes must be RNase-free to avoid degradation of viral RNA by RNases.
Automated viral RNA purification on the QIAcube
Purification of viral RNA using the QIAamp Viral RNA Mini Kit can be fully automated
on the QIAcube. The innovative QIAcube uses advanced technology to process
QIAGEN spin columns, enabling seamless integration of automated, low-throughput
sample prep into your laboratory workflow. The QIAcube performs the same steps as
the manual procedure (lyse, bind, wash, and elute) enabling you to continue using the
QIAamp Viral RNA Mini Kit for purification of high-quality viral RNA.
The QIAcube is preinstalled with protocols for purification of plasmid DNA, genomic
DNA, RNA, viral nucleic acids, and proteins, plus DNA and RNA cleanup. The range
of protocols available is continually expanding, and additional QIAGEN protocols
can be downloaded free of charge at www.qiagen.com/MyQIAcube.
Detailed protocols for using the QIAamp Viral RNA Mini Kit on the QIAcube are
provided with the QIAcube.
Determination of yield
Yields of viral RNA isolated from biological samples are normally less than 1 µg and
therefore difficult to determine photometrically. Keep in mind that the carrier RNA
(5.6 µg per 140 µl sample) will account for most of the RNA present. Quantitative
RT-PCR is recommended for determination of viral RNA yield.
Determination of viral RNA length
The size distribution of viral RNA purified using QIAamp spin columns can be checked
by denaturing agarose gel electrophoresis followed by hybridization with a virusspecific labeled probe and autoradiography (Sambrook, J. and Russell, D. W. [2001]
Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press).
QIAamp Viral RNA Mini Handbook 06/2012
11
Figure 1. Automated viral RNA purification. Viral RNA purification using the QIAamp Viral RNA Mini Kit can
be fully automated on the QIAcube.
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QIAamp Viral RNA Mini Handbook 06/2012
Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves,
and protective goggles. For more information, consult the appropriate safety data
sheets (SDSs), available from the product supplier.
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Ethanol (96–100%)*
1.5 ml microcentrifuge tubes
Sterile, RNase-free pipet tips (pipet tips with aerosol barriers for preventing crosscontamination are recommended)
Microcentrifuge (with rotor for 1.5 ml and 2 ml tubes)
For vacuum protocols
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QIAvac 24 Plus vacuum manifold (cat. no. 19413) or equivalent
VacConnectors (cat. no. 19407)
Vacuum Regulator (cat. 19530) for easy monitoring of vacuum pressures and
easy releasing of vacuum
Vacuum Pump (cat. no. 84010 [USA and Canada], 84000 [Japan], or 84020
[rest of world]) or equivalent pump capable of producing a vacuum of –800 to
–900 mbar
Optional: VacValves (cat. no. 19408)
Optional: QIAvac Connecting System (cat. no. 19419)
* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.
QIAamp Viral RNA Mini Handbook 06/2012
13
Important Notes
If preparing RNA for the first time please read “Handling RNA“ in the appendix of this
handbook (page 35). All steps of the QIAamp Viral RNA Mini protocols should be
performed quickly and at room temperature (15–25°C).
After collection and centrifugation, plasma (untreated or treated with anticoagulants other
than heparin) or serum can be stored at 2–8°C for up to 6 hours. For long-term storage,
freezing at –20°C to –80°C in aliquots is recommended. Frozen plasma or serum samples
must not be thawed more than once. Repeated freezing and thawing leads to denaturation
and precipitation of proteins, causing reduced viral titers and subsequently reduced yields
of the isolated viral RNA. In addition, cryoprecipitates formed by freeze–thawing will cause
clogging of the QIAamp membrane. If cryoprecipitates are visible, they can be pelleted by
briefly centrifuging at 6800 x g for 3 minutes. The cleared supernatant should be removed,
without disturbing the pellet, and processed immediately. This step will not reduce viral titers.
The QIAamp Viral RNA Mini procedure is not designed to separate RNA from DNA. To
avoid cellular DNA contamination follow the guidelines in ”Cellular DNA contamination”
on page 8 of this handbook.
The QIAamp Viral RNA Mini procedure isolates all RNA molecules larger than
200 nucleotides. Smaller RNA molecules will not bind quantitatively under the conditions
used.
Preparation of reagents
Addition of carrier RNA to Buffer AVL*
Add 310 µl Buffer AVE to the tube containing 310 µg lyophilized carrier RNA to obtain
a solution of 1 µg/µl. Dissolve the carrier RNA thoroughly, divide it into conveniently
sized aliquots, and store it at –20°C. Do not freeze–thaw the aliquots of carrier RNA
more than 3 times.
Check Buffer AVL for precipitate, and if necessary incubate at 80°C until the precipitate
is dissolved. Calculate the volume of Buffer AVL–carrier RNA mix needed per batch of
samples by selecting the number of samples to be simultaneously processed from Table 1.
For larger numbers of samples, volumes can be calculated using the following sample
calculation:
n x 0.56 ml = y ml
y ml x 10 µl/ml = z µl
where: n = number of samples to be processed simultaneously
y = calculated volume of Buffer AVL
z = volume of carrier RNA–Buffer AVE to add to Buffer AVL
Gently mix by inverting the tube 10 times. To avoid foaming, do not vortex.
* Contains chaotropic salt. Take appropriate laboratory safety measures, and wear gloves when handling.
Not compatible with disinfecting agents that contain bleach. See page 5 for safety information.
14
QIAamp Viral RNA Mini Handbook 06/2012
Table 1. Volumes of Buffer AVL and carrier RNA–Buffer AVE mix required for the QIAamp
Viral RNA Mini procedure
No.
samples
Vol. Buffer
AVL (ml)
Vol. carrier
RNA–AVE (µl)
No.
samples
Vol. Buffer
AVL (ml)
Vol. carrier
RNA–AVE (µl)
1
0.56
5.6
13
7.28
72.8
2
1.12
11.2
14
7.84
78.4
3
1.68
16.8
15
8.40
84.0
4
2.24
22.4
16
8.96
89.6
5
2.80
28.0
17
9.52
95.2
6
3.36
33.6
18
10.08
100.8
7
3.92
39.2
19
10.64
106.4
8
4.48
44.8
20
11.20
112.0
9
5.04
50.4
21
11.76
117.6
10
5.60
56.0
22
12.32
123.2
11
6.16
61.6
23
12.88
128.8
12
6.72
67.2
24
13.44
134.4
Note: The sample-preparation procedure is optimized for 5.6 µg of carrier RNA per
sample. If less carrier RNA has been shown to be better for your amplification system,
transfer only the required amount of dissolved carrier RNA to the tubes containing Buffer
AVL. (Use of less than 5.6 µg carrier RNA per sample must be validated for each particular sample type and downstream assay.)
Buffer AVL–carrier RNA should be prepared fresh, and is stable at 2–8°C for up to
48 hours. This solution develops a precipitate when stored at 2–8°C that must be redissolved by warming at 80°C before use. Do not warm Buffer AVL–carrier RNA solution
more than 6 times. Do not incubate at 80°C for more than 5 minutes. Frequent warming and extended incubation will cause degradation of carrier RNA, leading to reduced
recovery of viral RNA and eventually false negative RT-PCR results. This is particularly
the case with low-titer samples.
QIAamp Viral RNA Mini Handbook 06/2012
15
Buffer AW1*
Buffer AW1 is supplied as a concentrate. Before using for the first time, add the
appropriate amount of ethanol (96–100%) as indicated on the bottle and in Table 2.
Buffer AW1 is stable for 1 year when stored closed at room temperature (15–25°C),
but only until the kit expiration date.
Table 2. Preparation of Buffer AW1
Kit cat. no.
No. of preps
AW1 concentrate
Ethanol
Final volume
52904
50
19 ml
25 ml
44 ml
52906
250
98 ml
130 ml
228 ml
Buffer AW2†
Buffer AW2 is supplied as a concentrate. Before using for the first time, add the
appropriate amount of ethanol (96–100%) to Buffer AW2 concentrate as indicated on
the bottle and in Table 3.
Buffer AW2 is stable for 1 year when stored closed at room temperature (15–25°C),
but only until the kit expiration date.
Table 3. Preparation of Buffer AW2
Kit cat. no.
No. of preps
AW2 concentrate
Ethanol
Final volume
52904
50
13 ml
30 ml
43 ml
52906
250
66 ml
160 ml
226 ml
* Contains chaotropic salt. Take appropriate laboratory safety measures, and wear gloves when handling.
Not compatible with disinfecting agents that contain bleach. See page 5 for safety information.
†
Contains sodium azide as a preservative.
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QIAamp Viral RNA Mini Handbook 06/2012
Handling of QIAamp Mini columns
Owing to the sensitivity of nucleic acid amplification technologies, the following
precautions are necessary when handling QIAamp Mini columns to avoid crosscontamination between sample preparations:
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Carefully apply the sample or solution to the QIAamp Mini column. Pipet the
sample into the QIAamp Mini column without wetting the rim of the column.
Change pipet tips between all liquid transfer steps. The use of aerosol-barrier tips
is recommended.
Avoid touching the QIAamp membrane with the pipet tip.
After all pulse-vortexing steps, briefly centrifuge 1.5 ml microcentrifuge tubes to
remove drops from the inside of the lid.
Wear gloves throughout the procedure. In case of contact between gloves and
sample, change gloves immediately.
Spin protocol
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Close the QIAamp Mini column before placing it in the microcentrifuge.
Centrifuge as described.
Remove the QIAamp Mini column and collection tube from the microcentrifuge.
Place the QIAamp Mini column in a new collection tube. Discard the filtrate and
the old collection tube. Please note that the filtrate may contain hazardous waste
and should be disposed of properly.
Open only one QIAamp Mini column at a time, and take care to avoid generating
aerosols.
For efficient parallel processing of multiple samples, it is recommended to fill a
rack with collection tubes to which the QIAamp Mini columns can be transferred
after centrifugation. Used collection tubes containing the filtrate can be
discarded, and the new collection tubes containing the QIAamp Mini columns
can be placed directly in the microcentrifuge.
Vacuum protocol on the QIAvac 24 Plus
The QIAvac 24 Plus is designed for fast and efficient vacuum processing of up to
24 QIAGEN spin columns in parallel. Samples and wash solutions are drawn through
the column membranes by vacuum instead of centrifugation, providing greater speed
and reduced hands-on time in purification procedures.
In combination with the QIAvac Connecting System (optional), the QIAvac 24 Plus can
be used as a flow-through system. The sample flow-through is collected in a separate
waste bottle.
For maintenance of the QIAvac 24 Plus, please refer to the handling guidelines in the
QIAvac 24 Plus Handbook.
QIAamp Viral RNA Mini Handbook 06/2012
17
Processing QIAamp Mini columns on the QIAvac 24 Plus
QIAamp Mini columns are processed on the QIAvac 24 Plus using VacConnectors and
reusable VacValves. VacValves (optional) are inserted directly into the luer slots of the
QIAvac 24 Plus manifold and ensure a steady flow rate, facilitating parallel processing
of samples of different natures (e.g., plasma and serum), volumes, or viscosities. They
should be used if sample flow rates differ significantly to ensure consistent vacuum.
VacConnectors are disposable connectors that fit between QIAamp Mini columns and
VacValves or between QIAamp Mini columns and the QIAvac 24 Plus manifold. They
prevent direct contact between the column and VacValve or QIAvac 24 Plus manifold
during purification, thereby avoiding any cross-contamination between samples.
VacConnectors are discarded after a single use.
Handling guidelines for the QIAvac 24 Plus
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Always place the QIAvac 24 Plus on a secure bench top or work area. If dropped,
the QIAvac 24 Plus manifold may crack.
Always store the QIAvac 24 Plus clean and dry. For cleaning procedures, see the
QIAvac 24 Plus Handbook.
The components of the QIAvac 24 Plus are not resistant to certain solvents
(Table 4). If these solvents are spilled on the unit, rinse it thoroughly with water.
To ensure consistent performance, do not apply silicone or vacuum grease to any
part of the QIAvac 24 Plus manifold.
Always use caution and wear safety glasses when working near a vacuum
manifold under pressure.
Contact QIAGEN Technical Services or your local distributor for information
concerning spare or replacement parts.
The vacuum pressure is the pressure differential between the inside of the vacuum
manifold and the atmosphere (standard atmospheric pressure 1013 millibar or
760 mm Hg) and can be measured using the QIAvac Connecting System or a
vacuum regulator (see Figure 1). The vacuum protocol requires a vacuum pump
capable of producing a vacuum of –800 to –900 mbar (e.g., QIAGEN's, Vacuum
Pump). Higher vacuum pressures must be avoided. Use of vacuum pressures
lower than recommended may reduce DNA yield and purity and increase the
frequency of clogged membranes.
QIAamp Viral RNA Mini Handbook 06/2012
Table 4. Chemical resistance properties of the QIAvac 24 Plus
Resistant to:
Acetic acid
Chaotropic salts
Chlorine bleach
Chromic acid
Hydrochloric acid
SDS
Sodium chloride
Sodium hydroxide
Tween® 20
Benzene
Chloroform
Ethers
Phenol
Toluene
Urea
Not resistant to:
Figure 2. Schematic diagram of the Vacuum Regulator.
QIAamp Viral RNA Mini Handbook 06/2012
19
Setup of the QIAvac 24 Plus vacuum manifold
1.
Connect the QIAvac 24 Plus to a vacuum source. If using the QIAvac Connecting
System, connect the system to the manifold and vacuum source as described in
Appendix A of the QIAvac 24 Plus Handbook.
2.
Recommended: Insert a VacValve into each luer slot of the QIAvac 24 Plus that is
to be used (see Figure 2).
VacValves should be used if flow rates of samples differ significantly to ensure
consistent vacuum.
3.
Insert a VacConnector into each VacValve (see Figure 2) or directly into each luer
slot of the QIAvac 24 Plus that is to be used. Close unused luer slots with luer
plugs or close the inserted VacValve.
Perform this step directly before starting the purification to avoid exposure of
VacConnectors to potential contaminants in the air.
4.
Place the QIAamp Mini columns into the VacConnectors on the manifold (see
Figure 2).
5.
For nucleic acid purification, follow the instructions in the vacuum protocol.
Discard the VacConnectors appropriately after use.
Leave the lid of the QIAamp Mini column open while applying vacuum.
Switch off the vacuum between steps to ensure that a consistent, even vacuum is
applied during processing. For faster vacuum release, a vacuum regulator should
be used (see Figure 1).
Note: Each VacValve can be closed individually when the sample is completely
drawn through the spin column, allowing parallel processing of samples of
different volumes or viscosities.
6.
After processing samples, clean the QIAvac 24 Plus (see “Cleaning and
Decontaminating the QIAvac 24 Plus” in the QIAvac 24 Plus Handbook).
Note: Buffers AVL and AW1 used in QIAamp Viral RNA Mini procedure are not
compatible with disinfecting agents containing bleach. See page 5 for safety
information.
20
QIAamp Viral RNA Mini Handbook 06/2012
5
4
3
4
3
2
2
1
12
11
10
9
8
7
6
6
5
4
24
3
23
2
EN
QIAG
22
1
21
20
19
18
17
16
15
14
13
1
Figure 3. Setting up the QIAvac 24 Plus with QIAamp Mini columns using VacValves and VacConnectors.
1.
2.
3.
4.
5.
6.
QIAvac 24 Plus vacuum manifold
Luer slot of the QIAvac 24 Plus
VacValve (optional)*
VacConnector*
QIAamp column
Luer slot closed with luer plug
* Must be purchased separately.
Centrifugation
QIAamp Mini columns will fit into most standard 1.5 ml or 2 ml microcentrifuge tubes.
Additional 2 ml collection tubes are available separately.
Centrifugation of QIAamp Mini columns is performed at 6000 x g (8000 rpm) to limit
centrifuge noise. Centrifugation at full speed will not affect RNA yield. Centrifugation
at lower speeds for lysate loading and the first wash step is also acceptable, provided
that the complete solution is transferred through the membrane. At the second wash
step centrifugation at full speed is strongly recommended.
All centrifugation steps are carried out at room temperature (15–25°C).
QIAamp Viral RNA Mini Handbook 06/2012
21
Spin Protocol
Protocol: Purification of Viral RNA (Spin Protocol)
This protocol is for purification of viral RNA from 140 µl plasma, serum, urine, cellculture media, or cell-free body fluids using a microcentrifuge. For automated
purification of viral RNA using the QIAamp Viral RNA Mini Kit on the QIAcube, refer
to the QIAcube User Manual and the relevant protocol sheet.
Larger starting volumes, up to 560 µl (in multiples of 140 µl), can be processed by
increasing the initial volumes proportionally and loading the QIAamp Mini column
multiple times, as described below in the protocol. Some samples with very low viral
titers should be concentrated before the purification procedure; see ”Protocol: Sample
Concentration“ (page 29).
Alternatively, larger sample volumes can be processed using one of the following kits,
which provide simultaneous purification of viral DNA and RNA.
I
I
I
QIAamp MinElute® Spin Kit*
QIAamp MinElute Vacuum Kit
QIAamp UltraSens® Virus Kit
200 µl
500 µl
1000 µl
Important points before starting
I
I
Read “Important Notes” (pages 14–21) before starting the protocol.
All centrifugation steps are carried out at room temperature (15–25°C).
Things to do before starting
I
I
I
I
Equilibrate samples to room temperature (15–25°C).
Equilibrate Buffer AVE to room temperature for elution in step 11.
Check that Buffer AW1 and Buffer AW2 have been prepared according to the
instructions on page 16.
Add carrier RNA reconstituted in Buffer AVE to Buffer AVL according to instructions
on page 14.
Procedure
1.
Pipet 560 µl of prepared Buffer AVL containing carrier RNA into a 1.5 ml
microcentrifuge tube.
If the sample volume is larger than 140 µl, increase the amount of Buffer
AVL–carrier RNA proportionally (e.g., a 280 µl sample will require 1120 µl
Buffer AVL–carrier RNA) and use a larger tube.
* Fully automatable on the QIAcube. See www.qiagen.com/MyQIAcube for protocols.
22
QIAamp Viral RNA Mini Handbook 06/2012
2.
Add 140 µl plasma, serum, urine, cell-culture supernatant, or cell-free body fluid
to the Buffer AVL–carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing
for 15 s.
3.
Incubate at room temperature (15–25°C) for 10 min.
Viral particle lysis is complete after lysis for 10 min at room temperature. Longer
incubation times have no effect on the yield or quality of the purified RNA.
Potentially infectious agents and RNases are inactivated in Buffer AVL.
4.
Briefly centrifuge the tube to remove drops from the inside of the lid.
5.
Add 560 µl of ethanol (96–100%) to the sample, and mix by pulse-vortexing for
15 s. After mixing, briefly centrifuge the tube to remove drops from inside the lid.
Only ethanol should be used since other alcohols may result in reduced RNA
yield and purity. Do not use denatured alcohol, which contains other substances
such as methanol or methylethylketone. If the sample volume is greater than
140 µl, increase the amount of ethanol proportionally (e.g., a 280 µl sample will
require 1120 µl of ethanol). To ensure efficient binding, it is essential that the
sample is mixed thoroughly with the ethanol to yield a homogeneous solution.
6.
Carefully apply 630 µl of the solution from step 5 to the QIAamp Mini column
(in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge
at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column into a clean
2 ml collection tube, and discard the tube containing the filtrate.
Close each spin column to avoid cross-contamination during centrifugation.
Centrifugation is performed at 6000 x g (8000 rpm) to limit microcentrifuge
noise. Centrifugation at full speed will not affect the yield or purity of the viral
RNA. If the solution has not completely passed through the membrane, centrifuge
again at a higher speed until all of the solution has passed through.
7.
Carefully open the QIAamp Mini column, and repeat step 6.
If the sample volume was greater than 140 µl, repeat this step until all of the
lysate has been loaded onto the spin column.
8.
Carefully open the QIAamp Mini column, and add 500 µl of Buffer AW1. Close
the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini
column in a clean 2 ml collection tube (provided), and discard the tube containing
the filtrate.
It is not necessary to increase the volume of Buffer AW1 even if the original
sample volume was larger than 140 µl.
QIAamp Viral RNA Mini Handbook 06/2012
23
Spin Protocol
To ensure efficient lysis, it is essential that the sample is mixed thoroughly with
Buffer AVL to yield a homogeneous solution. Frozen samples that have only been
thawed once can also be used.
Spin Protocol
9.
Carefully open the QIAamp Mini column, and add 500 µl of Buffer AW2. Close
the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. Continue
directly with step 11, or to eliminate any chance of possible Buffer AW2
carryover, perform step 10, and then continue with step 11.
Note: Residual Buffer AW2 in the eluate may cause problems in downstream
applications. Some centrifuge rotors may vibrate upon deceleration, resulting in
flow-through, containing Buffer AW2, contacting the QIAamp Mini column.
Removing the QIAamp Mini column and collection tube from the rotor may also
cause flow-through to come into contact with the QIAamp Mini column. In these
cases, the optional step 10 should be performed.
10. Recommended: Place the QIAamp Mini column in a new 2 ml collection tube (not
provided), and discard the old collection tube with the filtrate. Centrifuge at full
speed for 1 min.
11. Place the QIAamp Mini column in a clean 1.5 ml microcentrifuge tube (not
provided). Discard the old collection tube containing the filtrate. Carefully open
the QIAamp Mini column and add 60 µl of Buffer AVE equilibrated to room
temperature. Close the cap, and incubate at room temperature for 1 min.
Centrifuge at 6000 x g (8000 rpm) for 1 min.
A single elution with 60 µl of Buffer AVE is sufficient to elute at least 90% of
the viral RNA from the QIAamp Mini column. Performing a double elution using
2 x 40 µl of Buffer AVE will increase yield by up to 10%. Elution with volumes of
less than 30 µl will lead to reduced yields and will not increase the final
concentration of RNA in the eluate.
Viral RNA is stable for up to one year when stored at –20°C or –70°C.
24
QIAamp Viral RNA Mini Handbook 06/2012
Protocol: Purification of Viral RNA (Vacuum Protocol)
Alternatively, larger sample volumes can be processed using one of the following kits,
which provide simultaneous purification of viral DNA and RNA.
I
I
I
QIAamp MinElute Spin Kit*
200 µl
QIAamp MinElute Vacuum Kit
500 µl
QIAamp UltraSens Virus Kit
1000 µl
Important points before starting
I
I
Read “Important Notes” (pages 14–21) before starting the protocol.
All centrifugation steps are carried out at room temperature (15–25°C).
Things to do before starting
I
I
I
I
I
Equilibrate samples to room temperature (15–25°C).
Equilibrate Buffer AVE to room temperature for elution in step 10.
Check that Buffer AW1 and Buffer AW2 have been prepared according to the
instructions on page 16.
Add carrier RNA reconstituted in Buffer AVE to Buffer AVL according to instructions
on page 14.
For processing using VacConnectors and VacValves, set up the QIAvac 24 Plus
as described on page 20.
* Fully automatable on the QIAcube. See www.qiagen.com/MyQIAcube for protocols.
QIAamp Viral RNA Mini Handbook 06/2012
25
Vacuum Protocol
This protocol is for purification of viral RNA from 140 µl plasma, serum, urine, cellculture media, or cell-free body fluids using the QIAvac 24 Plus or equivalent vacuum
manifold. Larger starting volumes, up to 560 µl (in multiples of 140 µl), can be
processed by increasing the initial volumes proportionally and loading the QIAamp
Mini spin column multiple times, as described below in the protocol. Some samples
with very low viral titers should be concentrated before the purification procedure; see
”Protocol: Sample Concentration“ (page 29).
Procedure
1.
Pipet 560 µl of prepared Buffer AVL containing carrier RNA into a 1.5 ml
microcentrifuge tube.
Vacuum Protocol
If the sample volume is larger than 140 µl, increase the amount of Buffer
AVL–carrier RNA proportionally (e.g., a 280 µl sample will require 1120 µl
Buffer AVL–carrier RNA) and use a larger tube.
2.
Add 140 µl plasma, serum, urine, cell-culture supernatant, or cell-free body fluid
to the Buffer AVL–carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing
for 15 s.
To ensure efficient lysis, it is essential that the sample is mixed thoroughly with
Buffer AVL to yield a homogeneous solution. Frozen samples that have only been
thawed once can also be used.
3.
Incubate at room temperature (15–25°C) for 10 min.
Viral particle lysis is complete after lysis for 10 min at room temperature. Longer
incubation times have no effect on the yield or quality of the purified RNA.
Potentially infectious agents and RNases are inactivated in Buffer AVL.
4.
Briefly centrifuge the tube to remove drops from the inside of the lid.
5.
Add 560 µl of ethanol (96–100%) to the sample, and mix by pulse-vortexing for
15 s. After mixing, briefly centrifuge the tube to remove drops from inside the lid.
Insert a QIAamp Mini column into the VacConnector on the QIAvac 24 Plus
vacuum manifold.
Only ethanol should be used since other alcohols may result in reduced yield and
purity of the RNA. Do not use denatured alcohol, which contains other substances
such as methanol or methylethylketone. If the sample volume is greater than
140 µl, increase the amount of ethanol proportionally (e.g., a 280 µl sample will
require 1120 µl of ethanol). To ensure efficient binding, it is essential that the
sample is mixed thoroughly with the ethanol to yield a homogeneous solution.
The collection tube from the blister pack can be saved for the centrifugation in
step 14.
6.
Make sure that the main vacuum valve (between the vacuum pump and the
vacuum manifold) and the screw cap valve (on the end of the QIAvac 24 Plus
vacuum manifold) are closed. Switch on the vacuum pump by pressing the power
switch.
The vacuum is applied only to the connecting system (if used) and not to the
vacuum manifold.
Note: For fast and convenient release of the vacuum pressure, the QIAvac
Connecting System or the Vacuum Regulator should be used, see ordering
information, page 41.
26
QIAamp Viral RNA Mini Handbook 06/2012
Carefully apply 630 µl of the lysate from step 5 into the QIAamp Mini column
without wetting the rim. Avoid touching the QIAamp Mini column membrane with
the pipet tip.
8.
Open the main vacuum valve. Be sure to leave the lid of the QIAamp Mini column
open while applying vacuum. After all lysates have been drawn through the
QIAamp Mini column, close the main vacuum valve and open the screw cap valve
to vent the manifold. Close the screw cap valve after the vacuum is released from
the manifold.
After closing the main vacuum valve, the vacuum is applied only to the connecting
system (if used) and not the vacuum manifold.
If the lysates from individual samples have not completely passed through the
membrane despite the VacValves of all other QIAamp Mini columns being
closed, place the QIAamp Mini column into a clean 2 ml collection tube (not
provided), close the cap, and centrifuge at full speed for 3 min or until it has
completely passed through. Continue with step 7 of the spin protocol on
page 23. Additional collection tubes can be purchased separately, see ordering
information, page 40.
Centrifugation is performed at 6000 x g (8000 rpm) to limit microcentrifuge noise.
Centrifugation at full speed will not affect the yield or purity of viral RNA.
9.
Repeat steps 7 and 8.
If the sample volume was greater than 140 µl, repeat these steps until all of the
lysate has been drawn through the QIAamp Mini column.
10. Apply 750 µl of Buffer AW1 to the QIAamp Mini column without wetting the rim.
Avoid touching the QIAamp Mini column membrane with the pipet tip.
It is not necessary to increase the volume of Buffer AW1 even if the original
sample volume was larger than 140 µl.
11. Open the main vacuum valve. After all Buffer AW1 has been drawn through the
QIAamp Mini column, close the main vacuum valve and open the screw cap valve
to vent the manifold. Close the screw cap valve after the vacuum is released from
the manifold.
12. Apply 750 µl of Buffer AW2 to the QIAamp Mini column without wetting the rim.
Avoid touching the QIAamp Mini column membrane with the pipet tip. Leave the
lid of the column open.
13. Open the main vacuum valve. After all Buffer AW2 has been drawn through the
QIAamp Mini column, close the main vacuum valve and open the screw cap valve
to vent the manifold. Close the screw cap valve after the vacuum is released from
the manifold.
QIAamp Viral RNA Mini Handbook 06/2012
27
Vacuum Protocol
7.
Vacuum Protocol
14. Close the lid of the QIAamp Mini column. Remove it from the vacuum manifold
and discard the VacConnector. Place the QIAamp Mini spin column in a clean
2 ml collection tube saved from step 5, and centrifuge at full speed for 1 min to
dry the membrane completely.
15. Place the QIAamp Mini spin column into a clean 1.5 ml microcentrifuge tube (not
provided). Discard the collection tube containing the filtrate. Carefully open the
QIAamp Mini spin column. Add 60 µl of Buffer AVE equilibrated to room
temperature. Close the cap, and incubate at room temperature for 1 min.
Centrifuge at 6000 x g (8000 rpm) for 1 min.
A single elution with 60 µl of Buffer AVE is sufficient to elute at least 90% of the
viral RNA from the QIAamp Mini spin column. Performing a double elution using
2 x 40 µl of Buffer AVE will increase yield by up to 10%. Elution with volumes of
less than 30 µl will lead to reduced yields and will not increase the final concentration of RNA in the eluate.
Viral RNA is stable for up to one year when stored at –20°C or –70°C.
28
QIAamp Viral RNA Mini Handbook 06/2012
Protocol: Sample Concentration
Plasma, serum, urine, cerebrospinal fluid, bone marrow, and other body fluids often
have very low viral titers. In these cases, concentrating samples of up to 3.5 ml to a
final volume of 140 µl is recommended.
Important point before starting
I
Use centrifugal microconcentrators such as Centricon® 100 (Amicon: 2 ml, cat.
no. 4211), Microsep 100 (Filtron: 3.5 ml, cat. no. OD100C40), Ultrafree® CL
(Millipore: 2 ml, cat. no. UFC4 THK 25), or equivalent from other suppliers.
1.
Apply up to 3.5 ml of sample to the microconcentrator following the
manufacturer’s instructions.
2.
Centrifuge according to manufacturer’s instructions to a final volume of 140 µl.
Some samples, plasma in particular, may be difficult to concentrate to 140 µl due
to high viscosity. Centrifugation for up to 6 hours may be necessary.
3.
Pipet 140 µl of concentrated sample into a 1.5 ml microcentrifuge tube, and
follow the QIAamp Viral RNA Mini spin protocol on page 22.
QIAamp Viral RNA Mini Handbook 06/2012
29
Sample Concentration
Procedure
Protocol: Purification of Cellular, Bacterial, or Viral
DNA from Urine
Buffer AVL, used in the QIAamp Viral RNA Mini procedure, inactivates the numerous
unidentified PCR inhibitors found in urine. For isolation of cellular, bacterial, or viral
DNA from urine for use in PCR, the QIAamp Viral RNA Mini spin protocol (page 22)
is recommended.
Urine often contains very low numbers of cells, bacteria, or viruses. In these cases, we
recommend concentrating samples of up to 3.5 ml to a final volume of 140 µl, as
described below, before processing.
For purification of DNA from Gram-positive bacteria, please contact QIAGEN
Technical Services.
Important point before starting
DNA from Urine
I
Use centrifugal microconcentrators such as Centricon-100 (Amicon: 2 ml, cat. no.
4211), Microsep 100 (Filtron: 3.5 ml, cat. no. OD100C40), Ultrafree-CL
(Millipore: 2 ml, cat. no. UFC4 THK 25), or equivalent from other suppliers.
Procedure
1.
Apply up to 3.5 ml of sample to the microconcentrator following the
manufacturer’s instructions.
2.
Centrifuge according to manufacturer’s instructions to a final volume of 140 µl.
Some samples, plasma in particular, may be difficult to concentrate to 140 µl due
to high viscosity. Centrifugation for up to 6 hours may be necessary.
3.
Pipet 140 µl of concentrated sample into a 1.5 ml microcentrifuge tube, and
follow the QIAamp Viral RNA Mini spin protocol on page 22.
30
QIAamp Viral RNA Mini Handbook 06/2012
Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. For
more information, see also the Frequently Asked Questions page at our Technical Support
Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical
Services are always happy to answer any questions you may have about either the
information and protocols in this handbook or sample and assay technologies (for
contact information, see back cover or visit www.qiagen.com).
Comments and suggestions
Little or no RNA in the eluate
a)
Carrier RNA not added
to Buffer AVL
Reconstitute carrier RNA in Buffer AVE and mix with
Buffer AVL as described on page 14. Repeat the
purification procedure with new samples
b)
Degraded carrier RNA
Carrier RNA reconstituted in Buffer AVE was not stored
at –20°C or underwent multiple freeze–thaw cycles.
Alternatively, Buffer AVL–carrier RNA mixture was
stored for more than 48 hours at 2–8°C. Prepare a
new tube of carrier RNA dissolved in Buffer AVE and
mix with Buffer AVL. Repeat the purification procedure
with new samples.
c)
Sample frozen and
Repeated freezing and thawing should be avoided.
thawed more than once Always use fresh samples or samples thawed only once.
d)
Low concentration
of virus in the sample
Concentrate the sample volume to 140 µl using a
microconcentrator. Repeat the RNA purification
procedure with a new sample. See “Protocol: Sample
Concentration” on page 29.
e)
Inefficient protein
denaturation in Buffer
AVL
Precipitate, formed in Buffer AVL–carrier RNA after
storage at 2–8°C, was not redissolved by heating
before starting the procedure. Redissolve the precipitate,
and repeat the procedure with a new sample.
f)
Buffer AVL prepared
incorrectly
Check Buffer AVL for precipitate. Dissolve precipitate
by incubation at 80°C.
g)
No ethanol added
to the lysate (step 5)
Repeat the purification procedure with a new sample.
h)
Low percentage ethanol Repeat the purification procedure with a new sample.
used
Use 96–100% ethanol in step 5. Do not use denatured
alcohol, which contains other substances such as
methanol or methylethylketone.
QIAamp Viral RNA Mini Handbook 06/2012
31
Comments and suggestions
i)
Isopropanol used
instead of ethanol
We strictly recommend the use of ethanol as
isopropanol causes reduced yields.
j)
RNA degraded
Often RNA is degraded by RNases in the starting
material (plasma, serum, body fluids). Ensure that the
samples are processed quickly. If necessary, add
RNase inhibitor to the sample. Check for RNase
contamination of buffers and water, and ensure that no
RNase is introduced during the procedure.
k)
RNase contamination
in Buffer AVE
Discard contaminated Buffer AVE. Repeat the
purification procedure with a new sample and a fresh
tube of Buffer AVE. Additional Buffer AVE is available
separately; see page 40 for ordering information.
l)
Buffer AW1 or AW2
prepared incorrectly
Check that Buffer AW1 and AW2 concentrates were
diluted with correct volumes of pure (96–100%)
ethanol. Do not use denatured alcohol, which
contains other substances such as methanol or
methylethylketone. Repeat the purification procedure
with a new sample.
m)
Buffer AW1 or AW2
prepared with 70%
ethanol
Check that Buffer AW1 and AW2 concentrates were
diluted with 96–100% ethanol. Repeat the purification
procedure with a new sample.
n)
Buffers AW1 and AW2 Ensure that Buffer AW1 and Buffer AW2 are used in
used in the wrong order the correct order in the protocol. Repeat the purification
procedure with a new sample.
RNA does not perform well in subsequent enzymatic reactions
a)
Little or no RNA in the
eluate
Check “Little or no RNA in the eluate,” above, for
possible reasons.
b)
Inefficient virus lysis in
Buffer AVL
Precipitate formed in Buffer AVL–carrier RNA due to
temperature change before start of process. Repeat the
procedure with new samples, and ensure that no
precipitate has formed in Buffer AVL–carrier RNA at
the beginning of the process.
c)
Buffer AVL prepared
incorrectly
Ensure that carrier RNA has been reconstituted in
Buffer AVE and added to Buffer AVL (see page 14).
d)
Too much carrier RNA
in the eluate
Determine the maximum amount of carrier RNA
suitable for your RT-PCR. Adjust the concentration of
carrier RNA added to Buffer AVL accordingly.
32
QIAamp Viral RNA Mini Handbook 06/2012
Comments and suggestions
e)
Reduced sensitivity
Determine the maximum volume of eluate suitable for
your RT-PCR. Reduce the volume of eluate added to the
RT-PCR.
f)
Buffers AW1 and AW2 Ensure that Buffer AW1 and Buffer AW2 are used in
used in the wrong order the correct order in the protocol. Repeat the purification
procedure with a new sample.
g)
New combination of
reverse transcriptase
and Taq DNA
polymerase used
If enzymes are changed, it may be necessary to
readjust the amount of carrier RNA solution added to
Buffer AVL.
DNA contamination
DNA and RNA present
in the sample
To avoid copurification of DNA, use of cell-free body
fluids for preparation of viral RNA is recommended.
Samples containing cells, such as cerebrospinal fluid,
bone marrow, urine, and most swabs, should be
made cell-free by centrifugation or filtration. If using
centrifugation, pellet the cells for 10 min at 1500 x g
and use supernatant for isolation of viral RNA. If DNAfree RNA is required, digest either the sample or the
eluate with RNase-free DNase. DNase in the eluate
must be inactivated by heat treatment (15 min, 70°C).
General handling
a)
Lysate not completely
passed through the
membrane
Using the spin protocol: Centrifuge for 1 min at full speed
or until all the lysate has passed through the membrane.
Using the vacuum protocol: Insufficient vacuum was
applied or the lid of the spin column was closed during
the vacuum step. Increase the vacuum, and open the
lid while applying the vacuum. If the vacuum pressure
cannot be increased, place the QIAamp Mini column
in a clean 2 ml collection tube, close the cap, and
centrifuge at 6000 x g (8000 rpm) for 3 min or until
the lysate has completely passed through the
membrane. Place the QIAamp Mini column into
another clean 2 ml collection tube, and discard the
tube containing the filtrate. Continue with step 7 of the
spin protocol on page 23.
QIAamp Viral RNA Mini Handbook 06/2012
33
Comments and suggestions
b)
Clogged membrane
Cryoprecipitates have formed in plasma due to
repeated freezing and thawing. Do not use plasma
that has been frozen and thawed more than once.
c)
Cross-contamination
between samples
To avoid cross-contamination when handling QIAamp
Mini spin columns, follow the guidelines in “Handling
of QIAamp Mini columns” on page 17. Repeat the
purification procedure with new samples.
d)
Vacuum pressure
too high/too low
Using a vacuum pressure that is too high may damage
the QIAamp membrane. Using too low a vacuum
pressure may cause reduced RNA yield and purity.
Use a vacuum regulator (see ordering information on
page 41) to adjust the pressure to –800 to –900 mbar
for all vacuum steps.
34
QIAamp Viral RNA Mini Handbook 06/2012
Appendix
Handling RNA
Ribonucleases (RNases) are very stable and active enzymes that generally do not
require cofactors to function. Since RNases are difficult to inactivate and only minute
amounts are sufficient to destroy RNA, do not use any plasticware or glassware
without first eliminating possible RNase contamination. Great care should be taken to
avoid inadvertently introducing RNases into the RNA sample during or after the
purification procedure. To create and maintain an RNase-free environment, the
following precautions must be taken during pretreatment and use of disposable and
non-disposable vessels and solutions while working with RNA.
General handling
Proper microbiological, aseptic technique should always be used when working with
RNA. Hands and dust particles may carry bacteria and molds and are the most
common sources of RNase contamination. Always wear latex or vinyl gloves while
handling reagents and RNA samples to prevent RNase contamination from the surface
of the skin or from dusty laboratory equipment. Change gloves frequently and keep
tubes closed whenever possible. During the procedure, work quickly to avoid
degradation of RNA by endogenous or residual RNases.
Disposable plasticware
The use of sterile, disposable polypropylene tubes is recommended throughout the
procedure. These tubes are generally RNase-free and do not require pretreatment to
inactivate RNases.
Non-disposable plasticware
Non-disposable plasticware should be treated before use to ensure that it is RNasefree. Plasticware should be thoroughly rinsed with 0.1 M NaOH,* 1 mM EDTA*
followed by RNase-free water (see “Solutions”, page 36). Alternatively, chloroformresistant plasticware can be rinsed with chloroform* to inactivate RNases.
Glassware
Glassware should be treated before use to ensure that it is RNase-free. Glassware used
for RNA work should be cleaned with detergent,* thoroughly rinsed, and oven baked
at >240°C for four or more hours (overnight, if more convenient) before use.
Autoclaving alone will not fully inactivate many RNases. Oven baking will inactivate
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
QIAamp Viral RNA Mini Handbook 06/2012
35
ribonucleases. Alternatively, glassware can be treated with DEPC* (diethyl
pyrocarbonate). Rinse the glassware with 0.1% DEPC (0.1% in water) overnight
(12 hours) at 37°C, and then autoclave or heat to 100°C for 15 minutes to remove
residual DEPC.
Note: Corex® tubes should be rendered RNase-free by treatment with DEPC and not
by baking. This will reduce the failure rate of this type of tube during centrifugation.
Electrophoresis tanks
Electrophoresis tanks should be cleaned with detergent solution (e.g. 0.5% SDS),*
rinsed with water, dried with ethanol,*† and then filled with a solution of 3% H2O2.*
After 10 minutes at room temperature (15–25°C), the electrophoresis tanks should be
rinsed thoroughly with RNase-free water.
Solutions
Solutions (water and other solutions)* should be treated with 0.1% DEPC. DEPC will
react with primary amines and cannot be used directly to treat Tris buffers.* DEPC is
highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol
and CO . When preparing Tris buffers, treat water with DEPC first, and then dissolve
Tris to make the appropriate buffer.
2
DEPC is a strong, but not absolute, inhibitor of RNases. It is commonly used at a
concentration of 0.1% to inactivate RNases on glass or plasticware or to create
RNase-free solutions and water. DEPC inactivates RNases by covalent modification.
Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation.
Carbethoxylated RNA is translated with very low efficiency in cell-free systems.
However, its ability to form DNA:RNA or RNA:RNA hybrids is not seriously affected
unless a large fraction of the purine residues have been modified. Residual DEPC must
always be removed from solutions or vessels by autoclaving or heating to 100°C for
15 minutes.
Add 0.1 ml DEPC to 100 ml of the solution to be treated. Shake vigorously to bring
the DEPC into solution, or let the solution bake for 12 hours at 37°C. Autoclave for 15
minutes to remove any trace of DEPC. It may be desirable to test water sources for the
presence of contaminating RNases since many sources of distilled water are free of
RNase activity.
Note: QIAamp Viral RNA buffers are not rendered RNase-free by DEPC treatment and
are therefore free of any DEPC contamination.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
†
Plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the
supplier’s instructions.
36
QIAamp Viral RNA Mini Handbook 06/2012
Ordering Information
Product
Contents
Cat. no.
QIAamp Viral RNA Mini Kit
(50)
For 50 minipreps: 50 QIAamp Mini
Spin Columns, Carrier RNA, Buffers,
and Collection Tubes (2 ml)
52904
QIAamp Viral RNA Mini Kit
(250)
For 250 minipreps: 250 QIAamp Mini
Spin Columns, Carrier RNA, Buffers
and Collection Tubes (2 ml)
52906
QIAcube and QIAcube accessories — for fully automated sample
preparation using QIAGEN spin-column kits
QIAcube (110 V)*
QIAcube (230 V)†
Robotic workstation for automated
purification of DNA, RNA, or proteins
using QIAGEN spin-column kits,
1-year warranty on parts and labor
Warranty PLUS 2 Full,
QIAcube
3-year warranty, 48-hour (2 working
9240834
days) priority response, all labor, travel,
and repair parts
Starter Pack, QIAcube
Pack includes: reagent bottle racks (3);
rack labeling strips (8); 200 µl filter-tips
(1024); 1000 µl filter-tips (1024);
1000 µl filter-tips, wide-bore (1024);
30 ml reagent bottles (18); rotor
adapters (240); rotor adapter holder
990395
Filter-Tips, 1000 µl (1024)
Sterile, Disposable Filter-Tips (racked)
990352
Rotor Adapters (10 x 24)
For 240 preps: 240 Disposable Rotor
Adapters; for use with the QIAcube
990394
QIAamp Viral RNA Mini
Accessory Set
Additional Buffers and Reagents;
1048147
for use with at least 11 x QIAamp Viral
RNA Mini Kits (50), catalog number
52904, or 5 x QIAamp Viral RNA Mini
Kits (250), catalog number 52906,
on the QIAcube
9001292*
9001293†
* US, Canada, and Japan.
†
Rest of world.
QIAamp Viral RNA Mini Handbook 06/2012
37
Ordering Information
Product
Contents
Cat. no.
Related products
QIAamp Virus Kits — for simultaneous purification of viral RNA
and DNA from plasma and serum
QIAamp MinElute Virus
Spin Kit (50)*
For 50 minipreps: 50 QIAamp MinElute
Columns, QIAGEN Protease, Carrier
RNA, Buffers, Collection Tubes (2 ml)
57704
QIAamp MinElute Virus
Vacuum Kit (50)
For 50 minipreps: 50 QIAamp
MinElute Columns, QIAGEN Protease,
Carrier RNA, Buffers, Extension Tubes
(3 ml), Collection Tubes (1.5 ml)
57714
QIAamp UltraSens Virus Kit
(50)†
For 50 viral nucleic acid preps:
50 QIAamp Mini Spin Columns,
Proteinase K, Carrier RNA, Collection
Tubes (2 ml), Buffers
53704
QIAamp MinElute Media Kit — for purification of DNA from liquid media
QIAamp MinElute Media Kit
For 50 minipreps: 50 QIAamp MinElute
Columns, QIAGEN Proteinase K,
Carrier RNA, Buffers, Extension Tubes
(3 ml), Collection Tubes (1.5 ml)
57414
QIAamp Media MDx Kit — for automated purification of DNA
from liquid media using the BioRobot ® MDx workstation
QIAamp Media MDx Kit
(12)
For 12 x 96 preps: 12 QIAamp
96 Plates, Buffers, Proteinase K,
S-Blocks, Disposable Troughs, Racks
with Elution Microtubes CL (0.4 ml),
Carrier RNA, Top Elute Fluid, Caps,
Tape Pad
965752
QIAamp Virus BioRobot Kit — for automated, high-throughput
purification of viral RNA and DNA from plasma and serum
QIAamp Virus BioRobot
MDx Kit (12)
For 12 x 96 preps: 12 QIAamp
96 Plates, RNase-free Buffers,*
QIAGEN Protease, Elution Microtubes
CL, Caps, S-Blocks, Carrier RNA
965652
* Fully automatable on the QIAcube. See www.qiagen.com/MyQIAcube for protocols.
†
Other kit sizes are available; see www.qiagen.com.
* Wash buffers are labeled with bar codes, and expiration date is stated on the Q-card in the kit.
38
QIAamp Viral RNA Mini Handbook 06/2012
Ordering Information
Product
Contents
Cat. no.
EZ1® Virus Mini Kit v2.0 — for automated purification of viral nucleic acids
from 1–6 or 1–14 samples per run
EZ1 Virus Mini Kit v2.0 (48)
For 48 preps: Reagent Cartridges
(Virus Mini v2.0), Disposable Filter-Tips,
Disposable Tip-Holders, Sample
Tubes (2 ml), Elution Tubes (1.5 ml),
Buffer AVE, Carrier RNA
955134
EZ1 Virus Card v2.0
Preprogrammed card for EZ1 Virus v2.0 9017330
protocols
MagAttract® Virus Mini M48 Kit — for automated, simultaneous
purification of viral DNA and RNA from serum and plasma using the
BioRobot M48 workstation
MagAttract Virus Mini
M48 Kit (192)
For 192 virus nucleic acid preps:
MagAttract Suspension B and
RNase-Free Reagents and Buffers
App. Package, M48,
Infectious Disease
Software protocol package for
infectious disease applications,
v. 2.0, on the BioRobot M48
workstation
955336
9016145
QIAamp RNA Blood Mini Kit — for total RNA purification from blood
and body fluids
QIAamp RNA Blood
Mini Kit (50)
For 50 RNA preps: 50 QIAamp Mini
Spin Columns, 50 QIAshredder Spin
Columns, Collection Tubes (1.5 ml and
2 ml), RNase-free Reagents, and Buffers
52304
QIAamp DNA Blood Mini Kit — for genomic DNA purification from
blood and body fluids
QIAamp DNA Blood Mini Kit For 50 minipreps: 50 QIAamp Mini
(50)*†
Spin Columns, QIAGEN Protease,
Reagents, Buffers, and Collection
Tubes (2 ml)
51104
* Fully automatable on the QIAcube. See www.qiagen.com for protocols.
†
Other kit sizes are available; see www.qiagen.com.
QIAamp Viral RNA Mini Handbook 06/2012
39
Ordering Information
Product
Contents
Cat. no.
QIAamp 96 DNA Blood Kit — for high-throughput genomic DNA
purification from blood and body fluids
QIAamp 96 DNA Blood Kit
(4)*†
For 4 x 96 preps: 4 QIAamp 96 Plates,
QIAGEN Protease, Reagents, Buffers,
Lysis Plates, and Collection Vessels
51161
QIAamp DNA Mini Kit — for genomic DNA purification from tissue,
blood, and body fluids
QIAamp DNA Mini Kit (50)*‡ For 50 minipreps: 50 QIAamp Mini
Spin Columns, Proteinase K, Reagents,
Buffers, and Collection Tubes (2 ml)
51304
Accessories
Buffer AW1 (concentrate)
242 ml Wash Buffer 1 Concentrate
for 1000 preparations
19081
Buffer AW2 (concentrate)
324 ml Wash Buffer 2 Concentrate
for 1000 preparations
19072
Buffer AVL
5 x 31 ml Viral Lysis Buffer and
5 x 310 µg Carrier RNA for 250
preparations
19073
Buffer AL
216 ml for 1000 preparations
19075
Buffer ATL
200 ml Tissue Lysis Buffer for 1000
preparations
19076
Buffer AE
240 ml Elution Buffer for 1000
preparations
19077
Buffer AVE (108 x 2 ml)
1020953
Carrier RNA (12 x 1350 µg)
1017647
Collection Tubes (2 ml)
1000 Collection Tubes (2 ml)
19201
QIAGEN Protease
125 mg (40–45 mAU/mg lyophilized)
19155
QIAGEN Protease
4 x 125 mg (40–45 mAU/mg
lyophilized)
19157
* Other kit sizes are available; see www.qiagen.com.
†
Requires use of the QIAGEN 96-Well-Plate Centrifugation System. Please inquire.
‡
Fully automatable on the QIAcube. See www.qiagen.com for protocols.
40
QIAamp Viral RNA Mini Handbook 06/2012
Ordering Information
Product
Contents
Protease Solvent
Cat. no.
1021055
QIAGEN Proteinase K (2)
2 ml (>600 mAU/ml, solution)
19131
QIAGEN Proteinase K (10)
10 ml (>600 mAU/ml, solution)
19133
QIAvac 24 Plus
Vacuum manifold for processing
1–24 spin columns: includes QIAvac
24 Plus Vacuum Manifold, Luer Plugs,
Quick Couplings
19413
VacConnectors (500)
500 disposable connectors for use
with QIAamp spin columns on luer
connectors
19407
VacValves (24)
24 valves for use with the QIAvac 24
and QIAvac 24 Plus
19408
Vacuum Pump
Universal vacuum pump (capacity
34 L/min, 8 mbar vacuum abs.)
QIAvac Connecting System
System to connect vacuum manifold
with vacuum pump: includes Tray,
Waste Bottles, Tubing, Couplings,
Valve, Gauge, 24 VacValves
19419
Vacuum Regulator
For use with QIAvac manifolds
19530
84000*
84010†
84020‡
For up-to-date licensing information and product-specific disclaimers, see the respective
QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are
available at www.qiagen.com or can be requested from QIAGEN Technical Services or
your local distributor.
* Japan.
†
US and Canada.
‡
Rest of world.
QIAamp Viral RNA Mini Handbook 06/2012
41
Notes
42
QIAamp Viral RNA Mini Handbook 06/2012
Trademarks:
QIAGEN®, QIAamp®, QIAcube®, BioRobot®, EZ1®, MagAttract®, MinElute®, UltraSens® (QIAGEN Group);
Centricon®, UltraFree® (Merck KGaA); Corex® (Corning, Inc.); Tween® (ICI Americas Inc.).
Limited License Agreement for QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit
Use of this product signifies the agreement of any purchaser or user of the product to the following terms:
1. The product may be used solely in accordance with the protocols provided with the product and this handbook
and for use with components contained in the kit only. QIAGEN grants no license under any of its intellectual
property to use or incorporate the enclosed components of this kit with any components not included within
this kit except as described in the protocols provided with the product, this handbook, and additional protocols
available at www.qiagen.com. Some of these additional protocols have been provided by QIAGEN users
for QIAGEN users. These protocols have not been thoroughly tested or optimized by QIAGEN. QIAGEN
neither guarantees them nor warrants that they do not infringe the rights of third-parties.
2. Other than expressly stated licenses, QIAGEN makes no warranty that this kit and/or its use(s) do not
infringe the rights of third-parties.
3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead
to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License
Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in
any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the
kit and/or its components.
For updated license terms, see www.qiagen.com.
© 1999–2012 QIAGEN, all rights reserved.
www.qiagen.com
Australia I [email protected]
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Belgium I [email protected]
Brazil I [email protected]
Canada I [email protected]
China I [email protected]
Denmark I [email protected]
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1072904 06/2012
Sample & Assay Technologies